The central focus of this proposal is to define the basic mechanisms of water/fluid transport in the lung and airways in health and disease, and to identify new targets for drug development. Aquaporins (AQPs) are water-transporting proteins expressed in lung microvasculature (AQP1), airways (AQP3 and AQP4), alveolar epithelium (AQP5) and submucosal gland epithelium (AQP5). In the initial award period, mice lacking each of the four major lung aquaporin water channels were generated and used to study the role of aquaporins in fluid transport in alveoli, large airways, and airway submucosal glands. An important finding - AQP5-dependent fluid secretion in airway submucosal glands - will be followed up in Aim 1. Novel aquaporin-selective inhibitors and model systems will be used to test the hypothesis that fluid secretion in human submucosal glands is regulated by AQP5 expression and function. Aim 2 will critically address the role of aquaporins in other aspects of lung physiology using inhibitors, mouse models, and human tissues. The hypothesis will be tested that lung/airway aquaporins facilitate fluid removal from the airspaces and lung parenchyma following clinically relevant disease/injury. In Aim 3, novel lung slice, cell culture and fluorescence methods will be used to define water and salt transporting mechanisms in distal airways, an important but under-studied epithelium. The hypothesis that AQP4 and CFTR are required for distal airway fluid absorption will be tested, and the role of the distal airway epithelium is lung fluid clearance will be determined. Together, these experiments will provide definitive information on the role of aquaporins in lung physiology and disease. A unique strength of our research program has been the introduction of new approaches to study lung physiology in vivo and in freshly harvested tissues, and the development of incisive biophysical tools (fluorescent probes, microscopy methods) and inhibitors for water and ion transport studies.